| Objective:Isoliquiritigenin (ISL), a simple chalcone-type flavonoid, which is derived from licorice compounds and mainly presents in roots of licorice and many other plants, foods, beverages and tobaccos. It has been reported that ISL possesses a wide range of potent biological and pharmacological activities including anti-oxidative, anti-tumor, anti-aging, anti-inflammatory, anti-diabetic activities and so on. It is easy to be chemically synthesized. Synthesis isoliquiritigenin (S-ISL) was used in this study. Our previous experiment showed that the inhibition concentration of S-ISL was the same as that of natural ISL. However, the induction of apoptosis, inhibition of invasion and migration effects of S-ISL on Tca8113 cells are not clear. Therefore, the aim of this study was to explore the mechanisms of S-ISL on Tca8113 cells including induction of apoptosis, inhibition of invasion and migration.Methods:S-ISL was synthesized and elucidated from its nuclear magnetic resonance spectrum and detected with high-performance liquid chromatography (HPLC). The cells viability was assessed using the Sulforhodamine B (SRB) assay. Tca8113 cells’cycle and apoptosis were detected by Flow cytometry (FCM). The double dye Annexin V-FITC/PI was used to distinguish from living cells, early and late apoptosis cells as well as necrotic cells. Agarose gel electrophoresis assay was used to detect the formation of DNA ladder.21,7’-dichlorfluorescein-diacetate (DCFH-DA), as fluorescent probe, was used to measure the levels of intracellular reactive oxygen species (ROS) which was induced by H2O2. Messenger RNAs for Bax and Bcl-2 were determined using RT-PCR. In order to clarify the underlying molecular mechanisms of anti-invasion and anti-migration of S-ISL, we took several methods including cell-matrix adhesion assay and modified Boyden Transwell chamber assay. The activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in Tca8113 cells were assessed through gelatin zymography assay. Messenger RNAs for PTEN, TIMP-2 and PAI-1 were determined using RT-PCR.Results:1. S-ISL induced Tca8113 cell apoptosisS-ISL inhibited Tca8113, HepG-2 and PC12 cells’ proliferation viability in a concentration-and time-dependent manner as compared with vehicle groups. The IC50 values of S-ISL were 17.70 μg/mL,10.04 μg/mL and 9.67 μg/mL, respectively, when Tca8113 cells were treated for 24 h,48 h and 72 h. The IC50 values were 19.07 μg/mL,15.08 μg/mL and 14.95 μg/mL, respectively, when HepG-2 cells were treated for 24 h,48 h and 72 h. And the IC50 values were 38.13 μg/mL,30.94 μg/mL and 26.85μg/mL, respectively after 24 h,48 h and 72 h treatment for PC12 cells. These results indicated that the inhibition efficiency of S-ISL on Tca8113 cells was much more potent than that of HepG-2 and PC 12 cells. Furthermore, S-ISL also induced Tc8113 cell apoptosis at a lower concentration while higher concentrations of S-ISL blocked the cells in G1 phase of mitosis by Flow Cytometry. Along with the increase of drug concentrations and times, the numbers of apoptotic cells were increased and DNA ladder were formed. Moreover, S-ISL also reduced intracellular ROS levels induced by H2O2. Compared with vehicle groups, there was down-regulation in gene expression levels of Bcl-2 while up-regulation in Bax in Tca8113 cells treated with S-ISL.2. S-ISL inhibited invasion and migration abilities of Tca8113 cellsCell-matrix adhesion assay showed that S-ISL significantly decreased adhesion abilities of Tca8113 cells on the matrigel-coated surface. The results of modified Boyden chamber assay showed that S-ISL markedly and concentration-dependently suppressed the invasion and migration activities of Tca8113 cells. The gelatin zymography assay suggested that S-ISL significantly induced the activities of MMP-2 and MMP-9. Moreover, S-ISL had a distinct regulation of PTEN, TIMP-2 and PAI-1 mRNA levels.Conclusion:In summary, our studies suggest that S-ISL has anti-tumor effects on Tca8113, HepG-2 and PC12 cells. Tca8113 cells are more sensitive to S-ISL. S-ISL can induce Tca8113 cells apoptosis and its mechanisms are attributed to the modulation of ROS contents and Bax and Bcl-2 gene expressions. S-ISL can inhibit invasion and migration of Tca8113 cells and its mechanism may be related to decrease of MMP-2 and MMP-9 via the modulation of PTEN, TEMP-2 and PAI-1 gene expressions. Therefore, the present study provides some experimental evidence that S-ISL may be of great value in searching for a potential cancer therapy against human tongue cancer. |
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